The present invention relates to methods for stimulating nerve cells, and more specifically, to methods for promoting attachment, proliferation and differentiation of nerve cells by electrical stimulation of the cells on electrically conducting polymers.
The development of methods for promoting the growth and differentiation of nerve cells has proved to be very difficult. Neurons have been found to have only a limited ability to regenerate. After about six months, most nerve cells lose their ability to reproduce, and the ability of damaged nerve cells to repair themselves is very limited. There also are few methods available for the stimulation of neuron extension and differentiation in vitro or in vivo.
Electrical charges have been found to play a role in enhancement of neurite extension in vitro and nerve regeneration in vivo. Examples of conditions that stimulate nerve regeneration include piezoelectric materials and electrets, exogenous DC electric fields, pulsed electromagnetic fields, and direct application of current across the regenerating nerve. Neurite outgrowth has been shown to be enhanced on piezoelectric materials such as poled polyvinylidinedifluoride (PVDF) (Aebischer et al., Brain Res., 436;165 (1987); and R. F. Valentini et al., Biomaterials, 13:183 (1992)) and electrets such as poled polytetrafluoroethylene (PTFE) (R. F. Valentini et al., Brain. Res. 480:300 (1989)). This effect has been attributed to the presence of transient surface charges in the material which appear when the material is subjected to minute mechanical stresses. Electromagnetic fields also have been shown to be important in neurite extension and regeneration of transected nerve ends. R. F. Valentini et al., Brain. Res., 480:300 (1989); J. M. Kerns et al., Neuroscience 40:93 (1991); M. J. Politis et al., J. Trauma, 28:1548 (1988); and B. F. Sisken et al., Brain. Res., 485:309 (1989). Surface charge density and substrate wettability have also been shown to affect nerve regeneration. Valentini et al., Brain Res., 480:300-304 (1989).
Neurites have been shown to preferentially migrate toward the cathode under steady electric fields. L. F. Jaffe and M. -M. Poo., J. Exp. Zool., 209:115 (1979); N. B. Patel and M. -M. Poo, J. Neurosc., 4:2939 (1984); and K. Shibib et al., Surg. Neurol., 29:372 (1988). Mechanisms for the observed effects which have been proposed include redistribution of cytoskeletal proteins such as actin (P. W. Luther et al., Nature, 303:61 (1983)), and other molecules (M. J. Politis et al., J. Trauma, 28:1548 (1988)), favorable protein conformational changes (R. F. Valentini et al., Biomaterials, 13:83 (1992)), and promotion of electrical communication between nerve stumps (K. Shibib et al., Surg. Neurol., 29:372 (1988)).
There are several drawbacks to these systems. In the case of the PVDF and PTFE systems, the polymers have to be poled (alignment of dipoles) for several hours above the glass transition temperature of the polymer in the presence of high electric fields of approximately 21 Kv. It is only after poling that these materials exhibit strong piezoelectric or electret behavior for finite lengths of time. In the systems which utilize application of electromagnetic fields (exogenous and in vivo) for neuronal stimulation, the applied field is not focused on neuronal tissue but rather broadly applied over the entire site of the injury.
Polypyrrole (PP) has been used in a matrix for controlled delivery of the neurotransmitter dopamine (L. L. Miller and Q. -X. Zhou, Macromolecules, 20:1594 (1987)) and as a biosensor for detection of glucose (L. D. Couves, Synt. Metals., 28:C761 (1989)) or other proteins (O. A. Sadik and G. G. Wallace, Analytica. Chimica. Acta., 279:209 (1993)). Cell-surface surface interactions and cellular functions have been shown to be controlled on PP thin films by either changing the oxidation state of the polymer (J. Y. Wong et al., Proc. Natl. Acad. Sci., USA., 91:3201 (1994)) or by changing the wettability of the polymer film using appropriate dopants (V. R. Shastri, Ph.D. Dissertation Rensselaer Polytechnic Institute, 1995).
There is a need for the development of materials for controlling nerve cell attachment, growth and regeneration, both in vitro and in vivo, which would permit in vitro cultivation of nerve cells over a prolonged period of time and manipulation of in vivo regeneration, differentiation and function of nerve cells.
It is therefore an object of the present invention to provide methods and compositions for stimulating the attachment and regeneration of cells including nerve cells in culture. It is another object of the present invention to provide methods and compositions for enhancing growth and regeneration of cells including nerve cells when implanted in vivo on artificial substrates and prostheses. It is a further object of the present invention to provide methods and compositions which can potentially be used to stimulate attachment and growth of nerve cells in vivo, thereby to permit repair of nerve damage, reconstruction of nerve tissue and replacement of lost nerve system function.